Self-contained microelectrochemical bioassay platforms and methods

ABSTRACT

Methods and devices for improved chemical and biological detection assays combined well defined microstructures having independently addressable electrodes with various surface immobilization electrochemical assays. Combining known chemical detection immobilization assays, electrochemically active moieties with microstructures having independently addressable electrodes provides for vastly improved methods of detecting microorganisms, chemical compounds, and measuring membrane transport.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/253,187, filed Sep. 24, 2002, and claims priority to U.S. patentapplication Ser. No. 09/978,734, now U.S. Pat. No. 6,887,714, filed Oct.15, 2001, and claims priority to U.S. Provisional Application Ser. No.60/240,691, filed Oct. 16, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a microvolume electrochemical detectionassay. More specifically, the present invention relates to microscalestructures having an analyte immobilizing surface and at least oneelectrode separate from the immobilizing surface. The microscale devicesare used in conjunction with an immobilization assay that utilizes anelectroactive complex to generate a current that may be detected by theelectrode. The small volumes and sensitivity of the assays used inconjunction with microstructures provides an extremely rapid detectionmethod having superior sensitivity to existing methods.

2. Prior Art

There is a great need to miniaturize the analytical methodologies andinstrumentation for making rapid and sensitive analyses in biological,environmental, medical and food applications. The interest in decreasingthe analysis volume is of value for samples which are precious,expensive, require high spatial chemical resolution, and need improvedthroughput (e.g. more sample capacity in a smaller space). This does notnecessarily demand better detection limits. However, improvements inlimits of detection increase sensitivity and accuracy and are alsobeneficial for dilute analyte in small samples.

One analytical methodology that has been successful at providing thehigh specificity and selectivity, and which is transferable to smallvolumes is the immunoassay. This approach has been primarily designedfor medical diagnostics, and combines the highly specific antigen(Ag)/antibody (Ab) interaction with the sensitivity of a transducersystem that may be optical, radiological, piezoelectric orelectrochemical. Immunoassays with electrochemical detection aredesirable for a wider range of uses because highly specific and precisecurrent measurements can be performed with simple instrumentation, usingopaque device materials, and in colored and turbid samples minimizingprior pre-treatment procedures.

Some of the smallest analyzed volumes have been reported for homogeneousimmunoassays, although with poorer detection limits than those of thepresent invention. One type of small homogeneous assay involves laserinduced fluorescence detection combined with fast separation of boundand unbound antibody-antigen complexes in microfluidic systems. Anotherhas the advantages of the simpler electrochemical detection and can beperformed in a drop (600 pL). The latter device, however, does notinclude a separation step, and therefore may be susceptible tointerferences from other sample species. Recently, an example of anelectrokinetically-driven microfluidic chip for heterogeneous bioassaysusing about 118 nL volume and assay times below 5 min was reported.Detection was performed with laser induced fluorescence. Complicationsinclude modifying the walls of the channel, which affects theelectroosmotic flow, and elution of fluorescent-labeled-immobilizedspecies to bring them to the detection site. Low detection limits (pg/mLor fM) were not possible.

Detection immediately adjacent to the surface-immobilizedimmunocomponents should supply the largest signals with the shortestincubation periods. This has been accomplished with scanningelectrochemical microscopy (SECM). Detection limits as low as 5.25 pg/mLhave been obtained. However, long total assay times were required (1 hand up). Only the sample volume is small (10's of pL up to several μL)which is spotted onto a surface (and dried, followed by rinsing) orimmobilized onto magnetic immunobeads first, which are subsequentlytransferred to a surface for electrochemical detection. Yet, theenzymatic generation of electrochemically-detected species is carriedout on large volumes that must keep both the SECM electrode and theauxiliary/reference electrodes in electrochemical contact. Consequently,this setup is not well-suited for integration with small volume handlingor automation and has added complexity due to the SECM instrumentationand operation.

Heterogeneous immunoassays for human serum albumin on a thick-filmelectrochemical device have been reported. The immunocomponents are alsoimmobilized adjacent to the detecting electrode and small volumes may beused at all stages of the immunoassays. However, unlike the presentinvention the immunocomponents are attached in a noncovalent fashion toall surfaces (instead of selected ones), the overall dimensions arelarge (several millimeters) and therefore, the volumes must be larger(30 μL) to cover electrodes and modified surfaces, the area of theimmuno active surface exposed to solution is less well defined becauseit depends upon the size of the drop and wetting properties of thesubstrate, and the detection (based upon potentiometric strippinganalysis) yields detection limits that are higher (0.2 pg/mL).

General immunoassay procedure involves immobilization of the primaryantibody (Ab, rat-anti mouse IgG), followed by exposure to a sequence ofsolutions containing the antigen (Ag, mouse IgG), the secondary antibodyconjugated to an enzyme label (AP−Ab, rat anti mouse IgG and alkalinephosphatase) (or a secondary antibody without a label, but followed by atertiary-antibody with a label and which binds to the secondary one),and enzyme substrate (e.g. p-aminophenyl phosphate; PAPp, where APconverts PAPp to p-aminophenol, PAPR, and the “R” is intended todistinguish the reduced form from the oxidized form, PAPo, thequinoneimine), which is electrochemically reversible at potentials thatdo not interfere with reduction of oxygen and water (e.g, at pH 9.0,where AP exhibits optimum activity). In addition, the enzyme-generatedelectroactive species (e.g., PAP_(R)) does not cause electrode fouling,unlike phenol whose precursor, phenylphosphate, is often used as theenzyme substrate. Although PAP_(R) specifically undergoes air and lightoxidation, these are easily prevented on small scales and short timeframes. Picomole detection limits for PAP_(R) and femtogram detectionlimits for IgG achieved in microelectrochemical immunoassays usingPAP_(p) volumes ranging from 20 μl to 360 μL have been reportedpreviously. In capillary immunoassays with electrochemical detection,the lowest detection limit reported thus far is 3000 molecules of mouseIgG using a volume of 70 μL and a 30 min or assay time. Those skilled inthe art will recognize the above described assay as a sandwich-typeimmunoassay and will appreciate that this is only one of manyimmunoassays. Alternatives include competitive binding immunoassays andimmunoassays utilizing a more general physisabsorbing material otherthan a primary antibody.

Immunoassays are only one category of a very wide variety of surfaceimmobilization chemical, biological and biochemical detection assays.Northern and southern blot assays are well known techniques fordetecting specific polynucleotide sequences. They involve surfaceimmobilization of polynucleotides. Surfaces having one or more lipidlayers may be used to immobilize and detect compounds having hydrophobicregions. Molecular interactions may also be taken advantage of todevelop surface immobilization chemical detection assays. When twomolecules are known to bind to one another, one may be covalentlyattached to a substrate. The substrate is then exposed to a sample suchthat the other interacting molecule is given an opportunity to bind tothe substrate bound molecule. The substrate is then rinsed leaving onlybound analyte on the substrate. A number of detecting methods may thenbe applied to the surface. Detecting methods include using secondaryantibodies as described above, detecting the bi-products of an enzymaticreaction characteristic of the analyte, spectroscopy, fluorescent,electrochemical analysis or other methods known to those skilled in theart.

These assays generally require a laboratory setting. A person wishing toanalyze a sample with one of the above described assays most usuallysends the sample to a laboratory. Even while in a laboratory, manychemical detection assays take a relatively long period of time.

The disadvantages of immunofluorescence assays (IFA) include their lowrecovery efficiency, long processing times, the need for highly trainedanalysts and high cost. In addition, IFA detection often involves thetime consuming and skill intensive step of filtering and analyzing watersludge for analytes that have been labeled with a fluorescent antibody.It is also often difficult to distinguish microorganisms from debrisbound non-specifically by the antibodies. The procedure is expensive andoften takes days to complete.

Flow cytometry is a method used to detect parasitic contamination ofwater samples. Flow cytometry techniques can quantify microorganisms butinvolves much preparation, and time and require extremely expensiveequipment.

Numerous problems are associated with prior art methods of detectingmicroorganisms and biological molecules in water and environmentalsamples. In addition to those mentioned and the general lack of precise,reliable assays, prior art techniques generally require that samples betransferred to a laboratory or to another remote location for theconduct of the assay. Prior art techniques lack the requisitereliability, speed and sensitivity to accurately detect microorganismsand biological compounds in contaminated water samples.

It is crucial that specific, rapid and highly sensitive assays bedeveloped to detect microorganisms and toxins accurately and reliably.The known methods of enzyme immunoassays and immunofluorescence do notfulfill these requirements. The detection, viability or quantity ofmicroorganisms found in water or other environmental samples cannot bereliably determined using prior art methods. There is a need for routineepidemiological surveillance and environmental monitoring that can beconducted on site to provide early detection of the parasite.

It is therefore desirable to provide a method for rapid chemical,biological or biochemical detection of analytes.

It is also desirable to provide a highly sensitive method for detectinglow amounts of analyte in a very small amount of sample.

It is also desirable to provide a method for detecting an analyte in asmall sample having very high accuracy.

SUMMARY OF THE INVENTION

In the present invention, microstructures are formed by using chemicaland/or physical etching processes in combination with thermalevaporation and other layering techniques. Alternating layers ofinsulating and conducting materials are applied to either a solid orflexible substrate, forming a series of tubular electrodes. Thesubstrate may have preformed holes in order to form pores or may haveholes drilled through them after the formation of cavities in order toform pores. The alternating conducting layers serve as electrodes. Howdeep the wells or pores are depends on how many layers are applied tothe initial substrate.

The methods used to form these microstructures allow them to beextremely small. Micropores and microcavities may be formed that areless than a hundred micrometers wide. In fact they may be less than 10μm wide. The depth of the microstructures ranges anywhere from less than10 μm to over 100 μm. Microstructures are combined with known chemicaldetection assays. Surface immobilization assays are especially wellsuited for these microstructures, although any assays susceptible toelectrochemical detection may be combined with these microstructures.Surface immobilization assays are well known to those skilled in the artand include, but are not limited to, immunoassays, northern and southernblots, western blots and incorporation of proteins into lipid layers.Surface immobilization assays are especially advantageous for use inmicrostructures because the small size of the structures allows for avery short distance between the analyte being captured or recognized andthe electrodes being used for electrochemical detection. In addition,the short distance between the electrodes used for detecting the analytealso accelerates both detection and amplification by means of redoxcycling. Another benefit of combining surface immobilization assays withelectrochemical microstructures is that physisorption of materials usedfor surface immobilization may be regulated within the microstructure.By applying electrical currents to various electrodes, the location ofanalyte binding materials within the microstructure may be controlled.This allows a certain material, such as protein binding styrene orprimary antibodies, to bind to insulating layers or specific electrodeswhile preventing physisorption of these molecules to working electrodes.

These microstructures having surface immobilization assays incorporatedwithin them may be further modified by the formation of a lipidbi-layer. Organic compounds may be used to anchor a lipid bi-layer tothe rim of a microcavity or, alternatively, to one or both openings of amicropore.

The present invention provides a self-contained, microelectrochemicalheterogeneous immunosensor on the smallest volumes reported to date (1μL for the antigen, 1 μL for the secondary antibody-enzyme conjugate,and 200 nL for the electrochemically detected species) and takes lessthan 30 min to both complete the assembly of immunoassay components ontothe antibody-modified surface and detect enzymatically-generatedspecies. The invention demonstrates the advantage of the close proximityof unmodified electrodes to modified surfaces and their application inthe analysis of small volumes. Using a microcavity withindividually-addressable electrodes on a microfabricated chip, a primaryantibody is selectively and covalently attached at a gold, recessedmicrodisk (RMD) at the bottom of the microcavity to the free end of selfassembled monolayers (SAM's). Non-specific adsorption to the surroundingmaterial, polyimide, of the microcavity device was eliminated.Electrochemical desorption was used to confine the immunoassays activityat the RMD. Alkaline phosphatase, conjugated to a secondary antibody, isused for the enzymatic conversion of the substrate p-aminophenylphosphate to p-aminophenol (PAP_(R)) and is detectable in less than 30 susing cyclic voltammetry at a ring or tubular nanoband electrode, whichis on the wall of the microcavity and immediately adjacent to themodified RMD. A third electrode, also within the region of themicrocavity, served as the counter/reference electrode. The invention issuitable for analysis with volumes down to 10 pL.

This self-contained, microelectrochemical enzyme-linked immunosorbentassay (ELISA) device that we report here has the advantages of the SECMsystems, but is better suited for small volumes, miniaturization, andfor integration with microfluidics (to improve ultra small volume samplehandling and speed). Thus, unlike electrochemical immunoassay techniquespreviously reported, the self-contained electrochemistry in the presentinvention eliminates the need for an external reference and auxiliaryelectrode. Because all electrodes are contained in the same small space,ultra small volumes are possible at all stages of the immunoassays. Inaddition, these devices offer the possibility of further redox cycling(leading to signal amplification) between detecting electrode and otherelectrodes. The fixed, close proximity between detector and modifiedsurfaces of microbeads or immunobeads makes low detection limitspossible and reproducible, and does not require micromanipulators. Theresponse is fast because of the short distance for enzymaticallygenerated species to diffuse from the RMD to the TNB. Finally,separation of the modified surface from the detecting electrode of theinvention has advantages over traditional electrochemical sensors wheredetecting electrodes are also the ones that are modified: (1) thestability of the modified surface is improved because there are noelectron transfer events through or changes in potential in that layer,and (2) it allows for a large electrochemical signal because thedetecting electrode is bare.

While standard sandwich-type ELISA's such as the one described above arevery useful because they are ubiquitous in the art of detection ofchemical or biological substances, other immunoassay methods are alsosuitable for use inside microcavities. Those skilled in the art willrecognize that there are a variety of immunoassay methods. Immunoassaysmay be used not only in sandwich-assays as described above, but alsocompetitive binding assays. Similarly, the primary antibody may bereplaced with a variety of chemical compounds. If the analyte is apolynucleotide, it may be desirable to use DNA as the primary probe. Theanalyte anneals to a segment of the DNA inside the microcavity and thesecondary probe will then bind to a segment the polynucleotide analyte.It is also known to utilize compounds that bind to proteins, lipids,carbohydrates, bacteria or viri in place of the primary antibody.Although using general compounds that bind to more general types ofmolecules will eliminate the specificity provided by the primary probe,the specificity of the overall immunoassay is preserved by use of thesecondary probe. Those skilled in the art will recognize that it iscommon practice to utilize compounds other than the primary probe forbinding of the analyte to the assay substrate.

In the present invention, a new immunoassay method has been developedthat is especially well suited for use in conjunction withmicrocavities. The above described methods of utilizing primaryantibodies or other compounds for primary probes or capture probes tobind the analyte to substrate may all be used in this newly developedassay. Immunoassays usually are enzyme-linked, thus providing the first2 letters of the acronym ELISA. The secondary antibody is linked to acatalytic protein, usually by either annealing the protein and antibodygenes together or by conjugating them in a chemical reaction. Theseenzymes conjugated to the secondary antibody react with their substrateto form a product which may then be detected. In the method describedabove, an enzyme converts PAPP to PAP_(r). PAP then cycles betweenelectrodes changing back and forth between the oxidized and reducedstate. This causes the voltametric signal to be amplified. The presentinvention includes the development of utilizing metal ion releasingcompounds attached to the secondary antibody in place of an enzyme. Thereleased metal ion then may be directly detected by the electrodeswithin the microcavity. Utilizing a metal releasing compound, such as ametal protein, provides a more reliable and more accurate immunoassaywhen used in conjunction with a microcavity.

In addition, both alkaline phosphatase and metal binding proteins(electroactive complexes) may both be used in conjunction withpolynucleotide hybridization assays, such as northern and southernblots. Electroactive complexes may be covalently attached to DNA or RNAprobes used in hybridization assays well known in the art. The probeswill bind to analyte DNA within the microcavity. The electroactivecomplexes may then produce a current that may be measured by theelectrodes within the microstructures. This is more accurate and fasterthan current methods using radioactive isotopes or fluorescingcompounds. In addition, it is much safer than the commonly usedradioactive isotopes.

The present invention also provides for the detection of microorganismwithin an aqueous solution. The primary antibodies specific for aparticular microbe may be used to immobilize a single microbe within amicrocavity. This allows for rapid electrochemical detection utilizing asecondary antibody having a covalently bound electroactive complex. Thisrapid detection of microbes is a significant advantage over currentexisting methodologies that often take 24 hours or longer to detectmicroorganisms.

Another significant improvement of the present invention is its abilityto determine whether or not detected microorganisms are alive or dead.Once microorganisms are detected using immnuoabsorbent techniques withina microstructure, they may be heat shocked. Heat shocking causesmicroorganisms to release polynucleotides. Hybridization assays eitherwithin the same microstructure or in one to which the analyte solutionis transferred may then be used to detect the released polynucleotides.Dead microorganisms do not release polynucleotides specific for theproduction of heat shock proteins when heated. Assays that are capableof determining not only the presence, but also viability ofmicroorganisms are especially useful in water treatment facilities.

Because the microstructures are so small, multiple, individuallyaddressable microstructures may be formed in a single chip. When onlyone microstructure is used for an assay, such multi structure arrays maybe re-used as many times as there are microstructures. Arrays are alsouseful when detecting very low concentrations of analytes. The multiplemicrostructures may be used simultaneously. This method of samplingprovides for a highly accurate assay of analytes present in very lowconcentrations.

This invention makes smaller volumes no more difficult to analyze thanmacrovolumes because all of the electrodes are prefabricated within thesame small volume, thereby allowing self-contained electrochemistry tooccur. Not only can heterogeneous immunoassays in such electrochemicalsystems be applied to smaller sample volumes than previously possible,but they also offer better detection limits, sensitivity, and speedbecause of the close proximity of electrodes and modified surfaces. Inaddition, a new polyimide passivation protocol protects the detectingelectrodes until they are needed and prevents immuno activephysisorption to undesirable locations. These procedures allowfabrication of accurate and reliable immunosensor arrays. This procedureis also promising for modification in enclosed microfluidic deviceswhere photo patterning may not be convenient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a cross sectional diagram of a microcavity;

FIG. 2 shows a perspective diagram of a microcavity;

FIG. 3 shows a flow chart of the fabrication procedure utilized to formelectrodes in a micropore on a polyimide film;

FIG. 4 shows a diagram of a microcavity used in conjunction with asandwich type ELISA, where the secondary antibody is bound to alkalinephosphatase;

FIG. 5 shows a schematic diagram of a microcavity being used inconjunction with a sandwich type immunoassay;

FIG. 6 shows a schematic diagram of a microcavity being used inconjunction with a competitive binding immunoassay;

FIG. 7 shows a schematic diagram of a microcavity being used inconjunction with an immunoassay utilizing a protein adhesion layerinstead of a primary antibody;

FIG. 8 shows a schematic diagram of a micropore being used inconjunction with a sandwich type immunoassay;

FIG. 9 shows a schematic diagram of a microimmunobead being used inconjunction with a microcavity;

FIG. 10 shows a diagram of a microstructure used in conjunction with asandwich type immuno assay used to immobilize and detect amicroorganism;

FIG. 11A shows a microcavity used in conjunction with a DNAhybridization assay, wherein the probe has an electro active complexcovalently attached to it;

FIG. 11B shows a microcavity used in conjunction with an alternative DNAhybridization assay, wherein the secondary probe has an electroactivecomplex covalently attached to it.

FIG. 12A shows a schematic diagram of an array of microcavities;

FIG. 12B shows a schematic diagram of an array of independentlyaddressable microcavities.

FIG. 13. Demonstration of the necessity of SAMs in the activity of theimmunoassay components. CV responses at 25 mV/s using an external setupin 5 ml of 4 mM PAP_(p) in 0.1 M Tris buffer, after the modified Aumacrochip had been soaked in the solution for 24 h. Only Au macrochipscontaining the complete assembly (Ab+Ag+AP−Ab) with SAMs resulted in asignal that indicated formation of PAP_(R); MUOL+complete assembly(solid curve), MUA+complete assembly (dashed curve), and completeassembly without SAMs (dotted curve);

FIG. 14. Timed CV responses at 50 mV/s using a 200 nL drop of 4 mMPAP_(p) in 0.1 M Tris buffer at a self-contained microelectrochemicalimmunosensor containing MUOL+complete assembly with 100 ng/ml mouse IgG.About five seconds after the drop of 4 mM PAP_(p) was placed on top ofthe microcavity, CV was performed, which provided the initial response.A second response was obtained after 30 s from the time the drop wasplaced on top of the modified cavity that indicated a significantincrease from the initial response. Subsequent responses were taken at30 s intervals up to 6 min. (For clarity, not all data are shown.);

FIG. 15. Timed CV responses using a 0.5 L drop of 4 mM PAP_(p) (in Trisbuffer) from a self-contained microelectrochemical immunosensorcontaining MUOL+complete assembly with 100 ng/ml mouse IgG at 50 mV/s.About five seconds after the drop of 4 mM PAP_(p) was placed on top ofthe microcavity, a current reading was recorded at a scan rate of 50mV/s that served as the initial response. A second response was recordedafter 45 s that indicated a significant increase from the initialresponse. Subsequent responses were taken at 30 s intervals up to 6min.;

FIG. 16. Demonstration that immunoactivity occurs only at the modifieddisk in the microcavity and that polyimide-passivation is successful. CVresponses are of a self-contained microelectrochemical immunosensor (50ng/mL IgG) after 5 min in 500 nL 4 mM PAP_(p) IN 0.1 M Tris, pH 9.0,before (solid line) and after (dashed line) electrochemical removal ofthe modifying layer (MUOL+Ab+Ag+AP+Ab) at the RMD (50 mV/s, TNB=working,top layer Au=auxiliary/reference);

FIG. 17. The sensitivity of the microcavity toward detection of PAP_(R)was evaluated by establishing a calibration curve. CV at 50 mV/s wascarried out on PAP_(R) solutions (200 nL each) of differentconcentrations ranging from 5.00 mM to 3.98 mM that were placed top ofthe 50 m cavity. The calibration curve, which plots the plateau currentfor the average of two sets of experiments for each concentration ofPAP_(R) gave a linear curve with a PAP_(R) detection limit of 4.4 nM or880 fmol. (TNB=working, top layer Au=auxiliary/reference); and

FIG. 18. Calibration curve for mouse IgG concentrations ranging from 5to 100 ng/ml with a detection limit of 56 zmol. The current readings(after 2 min in 200 nL of 4 mM PAP_(p) in 0.1 M Tris, pH 9.0) weredivided by the current signal from 200 nL of 4 mM PAP_(R) in 0.1 M TrispH 9.0, (50 mV/s, TNB=working, top layer Au=auxiliary/reference) usingeach cavity prior to modification.

DETAILED DESCRIPTION OF THE INVENTION

“Microstructures” refers to microcavities and micropores havingalternating insulating and conducting layers where at least one of theconducting layers serves as a working electrode to detect currentproduced by electroactive complexes. They generally have circular crosssections, but may also be polygonal. They may be formed on rigid orflexible substrates.

“Microassay” means any of a variety of immobilization assays performedwithin a microstructure and detected by electrochemical methodsdescribed herein. These assays include, but are not limited to, sandwichtype and competitive binding immunoassays, including those utilizingmicroimmunobeads, polynucleotide hybridization assays, such as northernand southern blots, and protein binding assays.

“Analyte” means any chemical compound, biomolecule, bacteria, virus orportions thereof susceptible to immobilization assays includingimmunoassays and polynucleotide hybridization assays.

“Electroactive complex” refers to any protein or other molecule capableof producing an electric current when activated by any of a number ofcontrollable parameters. This includes both redox enzymes, capable ofeither oxidizing or reducing a substrate molecule, and metalloproteins,capable of releasing metal ions. Synthetic molecules, such as dendrimersthat bind several metal ions, are also included.

“Sample” means a composition that may or may not include an analyte. Asample may be either aqueous, dissolved in an organic solvent or a solidsample that may be dissolved in either water or an organic solvent.Prior to being applied to microassay structures, a solid sample willneed to be dissolved in a suitable solution capable of dissolving asuitable electrochemical species.

“Microassay structure” means a microstructure that has been adapted toperform a microassay.

“Primary analyte binding material” refers to any of a variety ofcompounds and biomolecules used in immobilization assays to bind one ormore analyte to a surface. These include but are not limited to primaryantibodies, lipid layers, protein binding materials such as styrene,polynucleotides and combinations thereof.

“Secondary Analyte Binding Material” is any material capable of bindingto an analyte and being covalently bound to an electroactive complex.This includes secondary antibodies as used in ELISA's and polynucleotideprobes as used in hybridization assays.

“Activating agent” means any change in a controllable parameter thatinduces an electroactive complex to generate a currentelectrochemically. This includes the addition of a redox substrate,change in pH, change in temperature, application of an electric charge,change in concentration of a particular molecule and other factors knownto those skilled in the art.

The present invention includes a novel surface immobilizationelectrochemical assay. The invention combines known surfaceimmobilization and molecular interaction techniques with a novelelectrochemical detection method in a microstructure. Any of a number ofanalyte binding materials are applied to the substrate of an assaystructure. A sample to be tested for a specific analyte is thenintroduced to the assay structure. Any analyte present is then placedunder the proper conditions in which it may bind to the analyte bindingmaterial. The assay structure is then rinsed to remove all materialsother than bound analyte and assay solution. A secondary analyte bindingmaterial is then added to the assay structure under conditions allowingit to bind to the bound analyte. The assay structure is then rinsedagain such that only the bound analyte and secondary analyte bindingmaterial remain on the assay structure. The assay structure is thenimmersed in either the same or a second assay solution. The secondaryanalyte binding material has a covalently bound electrochemically activemolecule. When activated, the electrochemically active molecule createsa current within the assay structure. After the secondary analytebinding material is given sufficient time to bind to any analytepresent, the assay structure is again rinsed to remove any secondaryanalyte binding material that is not bound analyte. Theelectrochemically active complex is then activated. If analyte ispresent, then a current will be created within the assay structure. Thestrength of the current is directly related to the amount of analytepresent.

Those skilled in the art will recognize that there are at least twocurrent detection methods that utilize a primary and secondary analytebinding material. Perhaps the most common is the “ELISA”, utilizing aprimary antibody and a secondary antibody. The difference between thepresent invention and the existing ELISA schemes is the presentinvention's rapid detection of an electrochemically active moleculewithin a self-contained microstructure having an array of wells or poreswith microelectrodes incorporated therein. Existing ELISA's utilize afluorescent tag, an isotope label or enzymes that act upon a substrateto cause a color change. These existing methods are substantially lessaccurate and more time intensive than the present invention.

Another known detection method is the hybridization assay. A DNA probeis attached to an assay structure that has a complementary segment tothe analyte or target DNA. A secondary probe labeled with an enzyme orany other reporter molecule is then added to the assay that also has acomplementary segment to a different section in the target DNA binds.These hybridization assays when performed in the conventional way aresubject to the same shortcomings as current ELISA assays. They are lessaccurate and more time intensive than the present invention.

Those skilled in the art will recognize that a variety ofelectrochemically active molecules will be suitable for the presentinvention. The present invention contemplates the use of redox enzymesas well as metalloproteins. A redox enzyme, such as alkaline phosphatasereduces a substrate. The reduced substrate is then oxidized by a workingelectrode, thereby creating a current. The substrate is again reduced bythe electrochemically active enzyme and cycles back the electrode. Thisredox cycling amplifies the current and allows for detection ofextremely low concentrations of analyte. Those skilled in the art willappreciate that alkaline phosphatase is only one of many suitable redoxenzymes. Any enzyme capable of reducing a substrate rapidly will be asuitable enzyme. In addition to redox enzymes, metaloproteins are alsoespecially well suited for the present invention.

Those skilled in the art will recognize that there are a wide variety ofboth proteins and organic compounds capable of binding to metal ions.There are a number of well studied metalloproteins capable of binding tovarious metals such as iron, zinc, magnesium and copper to name a few.The genes that encode for these proteins may be attached to a secondaryantibody in the same way that enzymes are attached to secondaryactivating antibodies. After the secondary antibody binds to the analyteand the microcavity is rinsed, the metalloprotein is treated so that itreleases the metal ion incorporated within it. The metal ion attached tothe metalloprotein may be released in a variety of ways, depending onthe protein used. Many metalloproteins release the incorporated metalion upon a change in pH. This may be accomplished either by addingbuffer to the solution or utilizing the electrodes within themicrocavity themselves to release protons in the solutionelectrolitically. Other metalloproteins release their metal ions uponreaction with a secondary compound that may be added to the immunoassaysubsequent to the rinsing step. Yet, other metalloproteins release theirmetal ions in the presence of chelating agents, such asethylenediaminetetraacetic acid (EDTA). Those skilled in the art willrecognize that these methods of coaxing metalloproteins to release theirincorporated metal ions are well known to those skilled in the art.

It may also be desirable to synthesize novel electroactive complexes toattach to the secondary probe. Those skilled in the art of proteinengineering will recognize that there a number of common amino acidsequences capable of binding to metal ions. A Few examples are zincfingers, cysteine loops, heme groups and the His X₃ His and His Pro PheHis sequences. These amino acid sequences may be added to existing,known polypeptides or may be incorporated into novel peptide sequencesand spliced onto secondary antibodies. So long as the electroactivecomplex is capable of forming a stable bond with a metal ion orelectroactive molecule and may be induced to release the metalion/electroactive molecule by the addition of a compound, change in pH,change in temperatures or other means easily adaptable for use withmicrocavity or micropore assays.

One of the advantages of using a metal binding electroactive complexesis that it provides a metal ion that easily cycles between electrodes asit undergoes reduction and oxidation or vice versa. This creates anautomatic redox cycling reaction. Because of this, amplification isinherent to the assay. This eliminates the need for additional chemicalamplification steps. Those skilled in the art will realize that thisgreatly simplifies chemical detection.

There are a variety of metalloproteins known to the field ofbiochemistry. These include metallthionin, ferritin, heme, dendrimer,and staph nuclease. Those skilled in the art will recognize that theseare only a very few of the many polypeptides capable of binding to metalions. When using one of these polypeptides as an electroactive complexit may be desirable to splice or covalently bind several copies of thecarrier species gene to the end of the secondary antibody gene. Thiswould form a polypeptide polymer tail on the secondary antibody andincrease the number of metal ions for use in the microimmunoassaysensor. Metalloproteins are especially well suited for redox cyclingamplification by means of a counter electrode.

Those skilled in the art will appreciate that there are a variety ofactivating agents. Which one is used will depend on the electroactivecomplex used. It is known that some metalloproteins release metal ionswhen the pH of the solution is altered. Other metalloproteins releasemetal ions when other parameters are changed. These parameters include,but are not limited to, change in temperature, addition of a substrateor ligands, application of an electrical current and application ofelectromagnetic radiation. Which metalloprotein and which activatingagents are utilized will depend on a variety of factors including, butnot limited to, the analyte being studied, the material utilized toimmobilize the analyte, reaction temperature, reaction pH and theconcentrations of various reagents and other ingredients in thesolution.

One of the significant advantages of the device disclosed herein is thefact that it makes use of microcavities and micropores. The small sizeof these microstructures, less than a hundred micrometers in diameter,results in the electrodes being very close to one another. The shortdistance between electrodes greatly increases both the speed at whichsensing occurs and the sensitivity of the device due to the restrictionof diffusion of the species to the bulk solution and to the smallelectrodes confined within the picoliter volume of the microcavity.Alternate layers of insulator and conductor are applied to a substrateto create the microcavities. The substrate may be either rigid as when asilica chip is used or flexible, as when a polyimide film is used.Chemical and physical etching processes and photolithography may be usedto etch a pattern into the alternating layers of conductor and insulatoronto a substrate. Those skilled in the art will recognize chemicaletching as a common process. The process of forming microstructures onrigid silica wafers is described in the detail below.

FIG. 1 shows a cross sectional diagram of a basic microcavity. Substrate32 may be any of a variety of materials such as glass, ceramic, silicaor polyimide film. Flexible substrates may be used to formmicrostructures on flexible surfaces while rigid substrates are used toform microstructures on rigid surfaces. Conducting layer 34 is thenapplied to substrate 32 using thermal evaporation, sputtering or othermethods known to those skilled in the art. Conducting layer 34 may begold, copper or other suitable conducting materials. Depending on thematerials used, it may be desirable to apply a chromium or any otheradhesion layer prior to applying conducting layer 34. Standardphotolythographic techniques are utilized to apply alternatinginsulating layers 46 with conducting layer 38 sandwiched between them.Final conducting layer 42 is applied to give structural support to themicrostructure. Top layer 42 may also double as a counter or referenceelectrode. Contact pads 36, 40 and 44 are used to apply current toconducting layers 34, 38 and 42 respectively.

FIG. 2 shows a perspective diagram of the microstructure shown inFIG. 1. In FIG. 2 it can be seen how conducting layer 38 forms a tubularnanoband electrode within the microcavity. Current photolythographic andetching techniques allow microcavity 30 to be less than 10 mM indiameter. FIGS. 1 and 2 show a circular microcavity. However, thoseskilled in the art will appreciate that a microcavity cross-section mayhave a variety of geometries, including any polygon desired. While thisembodiment shows three conducting and 2 insulating layers, those skilledin the art will also appreciate that additional layers may be added tothe microstructure so that as many insulating and conducting layers asis desired may be applied.

When desiring to form micropores on a flexible polyimide film, it isoften desirable to form a pore in a film prior to the layering andetching process. Pores in a polyimide film are formed by an excimerlaser. As the laser passes through the film, it slowly disperses and theresulting pore has a funnel shape. The average diameter of the porewhere the laser enters the film is 70 mm, and the average diameter wherethe laser exits the film is 30 mm.

A flow chart for the fabrication procedure is shown in FIG. 3. Thepolyimide film was cut to a workable size, put into a carrier to patternthe gold layer, and placed at a vertical angle in the thermal evaporatorto reduce the chances of shorting between gold layers. A 50μ chromiumfilm is applied to the polyimide film by thermal evaporation in order toform an adhesion layer. Next, a 1,500 angstrom gold film was depositedalso by thermal evaporation. The process is then repeated on theopposite side of the film.

When forming a microstructure around a pore in a polyimide film, thephotoresist from the etching process is itself used as the insulatinglayer. A 10 micrometer thick positive photo resist film is applied tothe polyimide film. When the positive photoresist is exposed to UVlight, it washes away in developer solution. If the photoresist iscovered to block the UV light, it will not develop away in the solution.After application of the photoresist to both sides of the film, one sideis exposed to UV light. This way, only the photoresist on the sidefacing the UV light and the photoresist within the pore itself developaway in solution. This leaves an insulating layer on the side of thefilm facing away from the UV light source. Thermal evaporation is usedto apply a subsequent conducting layer to the side having photoresistinsulator. This process is repeated to form as many alternatingconducting and insulating layers as is desired.

Whether a cavity or pore is more desirable will depend on theenvironment in which the microstructure is to be used. Similarly,whether a rigid or flexible microstructure is formed will also depend onthe purpose to which the microstructure is to be used. Pores may provemore desirable in devices that use flow through analysis. They may proveespecially suitable for microfluidic devices. Flexible micropores andmicrocavities may prove more suitable in medical or biological testingapplications.

FIG. 4 shows a diagram of a microcavity adapted for use with a sandwichtype ELISA microassay. Microcavity 50 is formed by first applyingconductive layer 54. Insulating layers 58 have been formed withconducting layers 60 sandwiched between them to form a tubular nanobandelectrode. Finally, conducting layer 62 is applied to add structure andto provide a reference/counterelectrode. Droplet 74 is a solution inwhich the immunoassay is performed. Any solvent is suitable for droplet74 so long as it does not denature the proteins and antibodies withinit. Primary antibodies 56 may be attached to recessed micro disk 55formed at the bottom of microcavity 50 by insulating layer 54. This maybe done by first attaching a self assembling monolayer not shown toconducting layer 54 and then covalently binding primary antibodies 56 tothe self assembled monolayer.

After primary antibodies 56 have been attached to recessed microdisk 55,a sample solution containing analyte 64 is applied to microcavity 50.After sufficient time under proper conditions has been allowed foranalyte 64 to bind to primary antibody 56, the microcavity 50 is rinsedto remove all of the sample solution except for bound analyte 64.Secondary antibodies 67 are then added to microcavity 50. Secondaryantibody 67 is comprised of two parts, and analyte 64 specific antibodyportion 66 and a covalently bound electroactive complex 68. In thisembodiment, electroactive complex 68 is a redox enzyme.

Once secondary antibodies 67 have been given time to bind to analyte 64,microcavity 50 is again rinsed to remove excess secondary antibody 67.An activating agent is then introduced to the microcavity. In thisembodiment, the activating agent is an oxidized form of a redox compound70. Redox enzyme 68 reduces the redox compound into its reduced form 72.The reduced redox compound 72 discharges an electron to conducting layer60 and returns to its oxidized state 70. Redox enzyme 68 then reducesthe compound again, causing the redox compound to cycle between reducedand oxidized states, thus amplifying the signal. The current introducedto electrode 60 may then be measured which in turn allows measurement ofthe amounted analyte from which the concentration of analyte in theoriginal sample solution may be calculated. Those skilled in the artwill appreciate that redox cycling may also be facilitated or enhancedby applying a positive charge to a second electrode such ascounter/reference electrode 62.

The close proximity of electrodes 60 to the surface on which the assayis performed greatly increases the rate of detection. The extremelysmall volume of microcavity 50 also contributes to a very fast reactionrate.

FIG. 5 shows an alternative embodiment of a microstructure adapted for asandwich type ELISA microassay. In this embodiment, the cavity waslayered onto solid substrate 122. A glass slide 106 covers microcavity100 so as to prevent evaporation. With extremely small volumes such asthese, evaporation can become a problem. Primary antibody 108 iscovalently bound to the self assembled monolayer 123. Analyte 110 isbound to primary antibody 108. Analyte 110 is also bound to secondaryantibody 112 which is covalently linked to metalloprotein 114. In thisembodiment, metalloprotein 114 is bound to four manganese ions. Afterthe second rinse to remove excess secondary antibody, metalloprotein 114is activated so as to release its four manganese ions. These ions thenbegin to shuttle electrons from counter electrode 102 to workingelectrode 104. This electron shuttling between electrodes results inredox cycling that amplifies the signal. By measuring the current, theconcentration of analyte may be calculated. The embodiment in FIG. 5illustrates the improvement this invention makes over standard enzymelinked immunoassays. By linking a secondary antibody to ametalloprotein, especially one binding to several metal ions, thesecondary antibody becomes uniquely adapted for electrochemicaldetection within a microstructure. The redox cycling induced in theembodiments shown in FIGS. 4 and 5 further improve the immunoassays byincreasing both sensitivity and accuracy of analyte detection.

While standard sandwich-type immunoassays are very useful because theyare ubiquitous in the art of chemical detection, other immunoassaysmethods may be more suitable for use inside microcavities. Those skilledin the art will recognize that there are a variety of immunoassaysmethods. Immunoassays may be used not only in sandwich-assays asdescribed above, but also competitive binding assays as shown in FIG. 6.

Here, microcavity 100 again has primary antibodies 108 covalently linkedto self assembled monolayer 123. After the sample has been applied tomicrocavity 100, the microcavity is then rinsed. Next, competitivebinder 120, covalently linked to electroactive complex 114, is added tomicrocavity 100. Competitive binder 120 binds to primary antibodies 108that do not have analyte 110 bound to them. The electroactive complex114 is then activated so as to release bound metal ions 116, which beginredox cycling between electrodes. With this type of assay, the weakerthe current, the stronger the initial analyte concentration.

The primary antibody may be replaced with a variety of chemicalcompounds. If the analyte is a polynucleotide, it may be desirable touse DNA in place of the antibody. The analyte will then anneal to theDNA inside the microcavity and the secondary antibody will then bind tothe polynucleotide analyte. It is also known to utilize compounds thatbind to proteins, lipids, carbohydrates, bacteria or viri in place ofthe primary antibody. Although using general compounds that bind to moregeneral types of molecules will eliminate the specificity provided bythe primary antibody, the specificity of the overall immunoassays ispreserved by use of the secondary antibody. Those skilled in the artwill recognize that it is common practice to utilize compounds otherthan the primary antibody for binding of the analyte to the assaysubstrate.

FIG. 7 shows another type of immunoassays well suited for use inmicrocavities. The microcavity is built upon substrate 156 which may beeither a solid silica substrate or a flexible polyimide substrate.Electrodes 152 and 154 are separated by insulating layers 153. In thebottom of microcavity 150 is a protein adhesive layer 158. Those skilledin the art will recognize that there are a variety of materials to whichall polypeptides adhere. In this immunoassays, the sample is applied tothe microcavity and analyte 166 binds to layer 158, as do other proteins160. Secondary antibodies 164 are bound to carrier species 168 that eachcontain a metal ion 170. Secondary antibodies 164 bind to analyte 166.Electroactive complexes 168 are then activated to release ions 170 thatcreate a current between electrodes 152 and 154. This type of assay issimilar to that found in FIG. 5, except that the substance attached tothe substrate binds several proteins and is not specific to one moleculeas primary antibody 108 is.

FIG. 8 shows immunoassays micropore 200. Substrate 202 may be either arigid silica wafer or a flexible polyimide film. Alternating layers ofinsulator 210 and electrodes 204 and 206 are applied according to themethods described herein. Final layer 208 is comprised of conductormaterial and provides stability to the microstructure. Primaryantibodies 212 are attached to the interior of the micropore. Thesolution to be tested may be run either over or through pore 200. Theimmunoassays utilizes the same as that found in FIG. 5, a sandwichimmunoassays. Analyte binds to primary antibodies 212, secondaryantibodies bind to analyte, and the electroactive complexes attached tothe secondary antibody is activated to release its ions and induce anelectrical current. Those skilled in the art will realize that any ofthe immunoassays described herein are as suitable for use in microporesas they are for use in microcavities.

As these microstructures are very small, evaporation becomes a seriousconsideration. It may therefore be desirable to cover the microcavity,thus preventing evaporation. It may also be desirable to suspend a filmor lipid bilayer across the top of a microcavity or micropore. Transportproteins may be inserted into lipid bi-layers and the analyte studiedmay be a transported compound. In this situation, the microsensingdevice described herein may be used to detect and measure the activityof various membrane transport proteins.

FIG. 9 illustrates use of a microcavity in conjunction with animmunobead. Use of immunobeads allows the reaction of binding theanalyte to the primary antibody to take place somewhere besides themicrostructure. After the analyte has bound to the primary antibodies onthe immunobead, the immunobead is then inserted into the microcavity andthe immunoassays is carried out to completion as described above. Thoseskilled in the art will appreciate that immunobeads may be used eitherwith sandwich or competitive binding immunoassays. Those skilled in theart will also realize that microbeads may be used in other types ofassays.

In FIG. 9, immunobead 302 is covered in primary antibodies 304 whichbind to analyte 306. Immunobead 302 is then inserted into microcavity300. Microcavity 300 may be lined with material that facilitatesphysiabsorbtion of microbeads. Also, magnetic fields may be applied inorder to coax microbeads into microcavities.

FIG. 10 illustrates the use of a microassay to detect a microorganism.In this embodiment, microbe 336 is immobilized within microcavity 330 bybinding to primary antibodies 334 which are attached to substrate 332.After microbe 336 has been immobilized within microcavity 330, secondaryantibodies 338 having covalently attached electroactive complexes 340are then applied. Secondary antibodies 338 bind to microbe 336 andelectroactive complexes 340 are then activated. This results in acurrent being discharged into electrode 342 which may then be measuredin order to calculate the concentration of microbes 336. Because only 1or 2 microbes would fit inside the smaller microcavities, arrays such asthose shown in FIG. 12 are especially useful for this type of microbedetection. Many water born pathogens, such as C. Parvum, E. Coli,cholera, anthrax and other pathogens known to those skilled in the art,may be quickly, easily and inexpensively detected using the presentinvention.

It is possible to use electroactive complexes in conjunction with assaysbesides immunoassays. For example, probes for Northern and Southern blotoligonucleotides may be bound to these electroactive complexes. The blotassay may then be performed within a microcavity. This not only providesfor very sensitive detection, it is much safer than standard Northernand Southern blots that utilize radioactive isotopes. Those skilled inthe art will see a great advantage to a faster, safer type ofpolynucleotide assay.

FIG. 11A illustrates a hybridization microassay performed within amicrostructure. Microcavity 360 has polynucleotide binding material 364attached to substrate 362. Polynucleotides 366 from a sample are appliedto microcavity 360 after being denatured. They then bind to DNA bindingmaterial 364. Those skilled in the art will appreciate that it issometimes desirable to denature nucleotides 366 after binding tonucleotide binding material 364. Next, probes 368 having covalentlyattached electroactive complexes 370 are applied to microcavity 360.Probes 368 anneal to portions of polynucleotides 366 that arecomplimentary to the sequences of probes 368. Microcavity 360 is thenrinsed of excess probe 368 and electroactive complexes 370 are thenactivated in order to generate a current in working electrode 372. Thiscurrent may be measured in order to calculate the concentration ofpolynucleotides that are complimentary to probes 368. Many microbesrelease specific mRNAs into surrounding solution. Therefore, these typesof hybridization assays may also be used to detect of presence ofmicrobes in aqueous or other solutions.

FIG. 11B illustrates an alternative hybridization assay for use in amicrostructure. Microcavity 380 has primary probes 382 bound inside it.When analyte solution possibly having analyte bacteria in it is heatshocked and the solution is placed in microcavity 380. Heat shockpolynucleotides will then partially bind to primary probes 382. Primaryprobes 382 are complimentary only to a portion of the heat shockpolynucleotides. This allows a secondary probe 386 to bind to anotherportion of the heat shocked polynucleotide. The secondary polynucleotidehas an electroactive complex 388. Once the secondary polynucleotide hasbeen given time to bind the heat shocked polynucleotide, theelectroactive complex is activated so as to generate a current. If thereare no heat shock polynucleotides present, the secondary probe hasnothing to bind to, and no current is produced.

The hybridization assay described in 11B is especially well suited fordetermining the viability of microorganisms in solution. It is possibleto first use a microstructure to determine whether or not certainmicroorganisms, such as C. parvum, are present in the solution beinganalyzed. Microstructures such, as those described in FIG. 10 are wellsuited for this initial detection. If this assay comes up positive,indicating that microorganisms are present, the analyte solution may beheat shocked and subsequently introduced to a second microstructurehaving a hybridization assay like the one in FIG. 11A or FIG. 11B inorder to determine whether or not the microorganisms are alive. Thistype of immunoassays-hybridization assay combination is especiallysuitable for use in water treatment facilities to detect dangerousmicroorganisms contaminating water supplies.

FIG. 12 illustrates an array of microcavities. Array 400 consists ofseveral microcavities 408. Electrodes 402 are parallel to one anotherand contact multiple microcavities. Electrodes 404 are situatedsimilarly to electrodes 402, but are perpendicular to electrodes 402.Electrodes 404 also contact multiple microcavities. Each microcavity maybe designed to have a separate assay within it. Array 400 allows severalanalytes to be tested for simultaneously. Array 400 may also be composedof microstructures having assays for the same analyte. Electrodes arefused together such that each microstructure simultaneously detects thesame analyte. Such an array is especially useful when the analyte ispresent at extremely low concentrations. Those skilled in the art willrecognize this array structure as a means of performing an assay havinga sampling technique. This exponentially increases the accuracy of theassay.

FIG. 12B shows a partial array 420 individually addressablemicrocavities 422. In this particular embodiment, bottom electrode 424serves as a recessed micro disk electrode on the bottom of microcavities422. Bottom electrodes 424 have contact pads 428 that allow each of themto be addressed individually. Upper electrode 426 forms a thin nanobandelectrode within the microcavity. Upper electrodes 426 have contact pads430 that allow them to be individually addressable. For simplicity,array 420 only shows relatively few microcavities. Additionally,insulating layers and a top conducting layer are not shown either. Anarray such as the one shown in FIG. 12B is especially useful forfacilities desiring to test for pathogens in drinking water and otherliquids. Such an array allows for the fabrication of many microcavitieshaving the same microassay within them. Because they are individuallyaddressable, the microstructures may be utilized one at a time. Thiswould allow a single array of individually addressable microstructuresto be used many times before it is discarded. In addition, it is arelatively simple matter to short the cavities together so that they maybe used simultaneously in order to improve the accuracy of themicroassay. Such an array may be utilized to detect the presence ofvarious pathogens. Upon detection, pathogens may be heat shocked andhybridization assays as described in 11A and 11B may be utilized to testfor heat shocked polynucleotides. Those skilled in the art willappreciate that, especially on larger scales, it may be desirable toutilize a polymerase chain reaction (PCR) to increase the amount of heatshock polynucleotides present in the sample with micro-scale assays andsamples having high concentrations of pathogens, (PCR) is not alwaysnecessary. Those skilled in the art will appreciate that the addition ofa PCR step to an assay design to detect the presence of and thenviability of pathogens as a relatively simple matter.

For reasons of costs and simplicity of manufacture, it may be desirableto utilize assay structures larger than the microstructures describedabove. Low temperature co-fired ceramics (LTCC) are inexpensive andeasily manufactured. Gold ink conducting layers are readily patteredonto LTCC chips. Channels, troughs, wells and pores are all readilymachined onto LTCC chips (as well as the microstructures describedabove) using whole punching or other techniques. The LTCC chips are thenlayered and co-fired together to form a single chip having severallayers. The ceramic assay structures created are generally from 100micrometers to 1 millimeter in diameter. Although larger than themicrostructures described above, they may be formed easily and quickly.The relatively low cost of LTCC chips make them preferable tomicrostructures in many instances.

The combination of electrochemical assays with microstructures is alsouniquely well suited for measuring membrane transport and diffusion. Afilm or lipid bilayer may be formed over the entrance into themicrostructure. Referring to FIGS. 1 and 2, a film may be laid acrossthe top of layer 42. Alternatively, lipids having organothiol groups maybe attached to layer 42 in order to begin “anchor” a lipid bilayermembrane across the top of the microcavity.

Upon formation of a film or membrane, the microstructure may then beexposed to any of a number of ions, both metal ions and charged organicmolecules. When these ions traverse the film or membrane, they willgenerate a charge within the microstructure that can be measured. Inthis way, diffusion across membranes may be measured accurately. Thepresent invention provides a superior method of measuring ion diffusionacross various membranes. Other methods of measuring diffusion acrossmembranes involve either electrodes that are a substantial distance fromthe membrane or electrodes that are actually attached to the membrane.Because the electrode in the present invention is only a few micrometersfrom the membrane, highly accurate membrane diffusion and transportmeasurements are possible. When electrodes are attached to the membranethemselves, this covalent attachment draws into question the accuracy ofsuch measurements. It is unknown how attachment of the electrode to themembranes truly affects these diffusion measurements. Those skilled inthe art will appreciate that the present invention is a vastly superiordevice for measuring ion diffusion and transport.

In the case of a lipid bilayer across a microstructure, it is alsopossible to incorporate membrane transport proteins into the lipidbilayer, thereby facilitating measurement of membrane transport by anyof a variety of ion transporting proteins. Currently, the most accurateway of measuring protein transport is by measuring degradation of ATPinto ADP. This was primarily accomplished using ATP having P³²incorporated as the tertiary phosphorus. Those skilled in the art willappreciate that the method of the present invention is a much safermethod of measuring transport. Furthermore, this method obviously onlyapplies to ion transport proteins that are ATPase's. Passive transportproteins may not be measured by this method. Furthermore, measuring iontransport by ATPase's activity is often inaccurate due to the presenceof other non-ion transporting ATPase's in the membranes, such asFlippase's. Those skilled in the art will appreciate that the presentinvention prevents such interference in measurements.

EXAMPLE 1

Fabrication of macrochips. Au macrochips (approximately 1.4 cm×2 cm,where the electroactive area is about 0.6 to 1 cm₂) were made from a 125mm diameter silicon wafer substrate that had 1.4 to 1.8 m S_(i)O₂deposited on both sides at 250° C. by plasma enhanced chemical vapordeposition (PEVCD, Plasma Therm System VII). Deposition of a 15 μMadhesion layer of Cr and 1000 μm of Au was carried out using an EdwardsAuto 306 TURBO thermal evaporator (Edwards High Vacuum InstrumentInternational, West Sussex, UK). The Au macrochips were diced to size byhand using a diamond scribe. Polyimide (PI) macrochips were made usingthe Au macrochips as the starting substrate and spin coating a 4 μmthick layer of polyimide followed by cross-linking with UV light at 350nm for 12 s and curing at 150° C. for 30 min and at 250° C. for another30 min. The Au coated silicon wafer was spin-rinsed-dried (SRD) using ST270D (Semitool, Calif.) for a total of 400 s before spin coating the PI.This process completely covers the Au so that the metal does notinfluence subsequent surface-modification experiments.

Fabrication of microcavity devices. Devices containing functional 50-μmmicrocavities were fabricated where three patterned layers of Au (withcorresponding Cr adhesion layers) are separated from each other bylayers of PI on S_(i)O₂-coated silicon wafers. The microcavities areformed by reactive ion etching through these layers, which exposes a RMDelectrode at the bottom (50 μm in diameter and 8 μm deep), a TNBelectrode along the wall of the microcavity (˜500 μm wide and 157 μmlong) and a top layer of gold at the rim. The microcavities were cleanedby sonicating (L&R PC3 Compact High Performance Ultrasonic CleaningSystem) in acetone or water followed by rinsing with DI water. The chipswere then dried with N₂ gas and stored in DI water until needed. In anelectrochemical study, electrode fouling in the microcavity can beeliminated by sonicating for 30 s in acetone or DI water and minimizedby keeping the microcavity in a vial of DI water when not in use.

Macrochip studies to passivate the polyimide insulator. The extent ofphysisorption of active immunoassays components on PI was studied onPI-coated Au, macrochips. Studies to eliminate physisorption involvedpre-testing PI macrochips with different chemical species. The PImacrochips were then carried through all of the same steps of theimmunoassays assembly both with or without SAMs of MUA or MUOL as thoseused to modify the Au macrochips (see assembly steps below). Theactivity of the modified PI surfaces to convert PAP_(p) to PAP_(R) wasevaluated by electrochemical detection of the PAP_(R) in the solutionsurrounding the PI surface.

Pretreatment of PI macrochips involved exposing the chips to the one ofthe following solutions overnight: acetate TBSA, acetate TBSA thenrinsed three times in DI water followed by exposure to 1.2 mM EDC for 10min and exposure to 4 mM butanol overnight, acetate TBSA then rinsedthree times in DI water followed by exposure to 1.2 mM EDC for 10 minand exposure to 4 mM propionic acid overnight, acetate TBSA then rinsedthree times in DI water followed by exposure to 1.2 mM EDC for 10 minand exposure to 4 mM butanol with 4 mM propionic acid overnight, 4 mM1-mercaptohexane, 4 mM diphenylamine (DPA), 4 mM MOD, 4 mM MOD inacetate TBSA, and 4 mM DPA in acetate TBSA. The 4 mM butanol, 4 mM DPA,4 mM mercaptohexane, and 4 mM MOD were prepared in ethanol that waspurged with Ar. The 4 mM propionic acid solution was prepared with DIwater. The PI macro chips were separately soaked overnight in each ofthe different solutions. The macro chips dipped in solutions prepared inethanol solvents were first rinsed three times with ethanol. Afterwards,all the chips were rinsed three times in DI water before exposure tosubsequent solutions.

Microcavity pretreatment to passivate PI surfaces and electrodecleaning. The best approach for passivating immunoactivity on PIinvolved pretreatment with 4 mM MOD in acetate TBSA in an Ar-filledglovebag before assembling the immunoassays components. Thispretreatment also passivates the gold surfaces of the microcavitydevice. Passivation was performed by exposing bare microcavity devicesto 5 mL acetate TBSA with 4 mM MOD overnight. These were rinsed threetimes with acetate TBSA and dried with Ar. The passivating films on theRMD (before immunoassays components were assembled) and on the TNB andtop layer gold (after immunoassays components were assembled) wereremoved by electrochemically cycling between +1.5 V to −0.5 V inelectrolyte solution of 1 mM C_(a)Cl₂ in 0.1 M KCl for at least 30 minat 30 V/s. An alternative procedure involved holding the potential at+0.7 V or −0.5 V in 1 mM C_(a)Cl₂ in 0.1 M KCl solution for at least 30min. Extent of electrode cleanliness and passivation was determined byCV in a solution of 4 mM K₃F_(e)(CN)₆, 1 mM C_(a)Cl₂, and 0.1 M KCl.

After electrochemical desorption, the microcavities were cleaned byrinsing with DI water. The chips were dried with N₂ gas and stored in DIwater until needed. The modified chips are refrigerated in acetate TBSA.

Determination of immunoassays activity on the silicon wafer. Todetermine whether physisorption to silicon occurs during the macrochipimmunoassays, silicon wafer pieces (approximately 1.4 cm×2 cm, withoutAu or PI) were subjected to the same surface modification processes usedfor the Au macrochips. The activity of the modified silicon wafermacrochips to convert PAP_(p) to PAP_(r) was evaluated byelectrochemical detection.

Self-assembled monolayers. The Au macrochips were cleaned in piranhasolution (30:70 (v/v) of 30% H₂O₂ and concentrated H₂SO₄) for 30 min andthoroughly rinsed for 30 min with running DI water before use. SAMpreparation, rinsing, and drying were carried out completely in anAr-purged glovebag after the cleaning step to eliminate oxidation ofSAMs by air (or ozone). The Au macrochips were soaked in solutions ofeither 4 mM MUA or 4 mM MUOL in Ar-purged ethanol for 24 h to form SAMs,followed by rinsing with Ar-purged ethanol three times in each of threeseparate test tubes inside the glovebag. The chips were dried with Arand kept in closed vials before use. The same procedure was followed forthe formation of SAMs on surfaces of the microcavity, with the exceptionthat the microcavity devices were cleaned by sonication in ethanol for30 s, instead of piranha solution.

Immobilization of the primary antibody. A working solution of 24 g/mL Aband 1.2 mM EDC in PB was prepared by combining appropriate volumes ofstock solutions of 1.3 mg/mL Ab and 2.4 M EDC in PB, followed bydilution with PB buffer. All of the following steps for antibodyimmobilization were performed inside a glove bag filled with Ar.SAM-modified macrochips and microcavity devices (which were pre-testedwith acetate TBSA and MOD, and for which passivation had beenelectrochemically removed from the RMD) were soaked in the Ab/EDCworking solution for 2 h (1 mL for the macrochip and 200 mL for themicrocavity inside a water-saturated, parafilm-sealed petri plate). TheEDC assists covalent attachment of the Ab to the free end of the SAMs43, 59, 60 The chips were rinsed with 1 M NaCl three times and thensoaked three times in acetate TBSA (1 mL for the macrochip for 15 minand 50 mL for the microcavity for 30 s, each time) to eliminatenon-specifically adsorbed Ab.

Capture of antigen, mouse IgG. Working solutions of the Ag were preparedby dilution of an 11.2 mg/ml Ag stock solution in 0.01 M sodiumphosphate buffer in 0.5 M NaCl (pH 8.0) with acetate TBSA.Ab-immobilized macrochips were exposed to a 1 mL solution of 100 ng/mLAg for 1 h and then rinsed three times with 1 mL of acetate TBSA for 15min. The Ab-immobilized microcavities were exposed to varyingconcentrations of Ag ranging from 5 ng/mL to 100 ng/mL by leaving a 1 mLdrop of the solution on top of the microcavity for 10 min in a watervapor-saturated petri dish sealed with parafilm (to minimizeevaporation). A 1 μL drop in this humid environment does not show anysignificant evaporation after 66 h at room temperature in thelaboratory. The microcavity was rinsed with 50 mL of acetate TBSA threetimes at 10 s each. These steps were performed outside of the glovebag.

Completing the immunoassay assembly with AP−Ab. A working solution of700 ng/mL AP−Ab was prepared by diluting a 0.7 mg/mL AP−Ab stocksolution in 0.01 M Tris-HCl in 0.25 M NaCl (pH 8.0) with acetate TBSA.Ab-immobilized macrochips that had been exposed to Ag and rinsed, weresubsequently exposed to 1 mL of the AP−Ab working solution for 3 h andthen rinsed by soaking three times in 5 mL of acetate TBSA for 15 mineach to eliminate non-specifically adsorbed AP−Ab. Ab-immobilizedmicrocavity devices that had been exposed to Ag and rinsed, were exposedto 1 L of the AP−Ab working solution for 10 min while inside a parafilmsealed water vapor-saturated petri dish and then rinsed three times with50 L acetate TBSA for 10 s each. These steps were performed outside ofthe glovebag.

After deposition of the SAMs with the complete assembly (Ab+Ag+AP−Ab) onthe microcavity devices, the passivation layers of the top layer Au andTNB were removed using the same procedures that were used for theremoval of the passivation at the RMD. The cleaned TNB and the top layerAu could then be used as working and combinationpseudoreference/auxiliary electrodes, respectively.

Enzymatic generation of PAP_(r). The enzyme substrate solution was 4 mMPAP_(p) in 0.1 M Tris at pH 9.0, as previously described. The solutionwas purged with Ar and kept from light to minimize oxidation.Macrochips, containing the complete immunoassay assembly, were rinsedthree times with 5 mL 0.1 M Tris at pH 9.0 at 10 min each before soakingin 5 mL of Ar-purged (15-30 minutes) PAP_(p) solution inside a sealedbeaker wrapped in aluminum foil for 24 h inside a glove bag filled withAr. Microcavity devices, containing the complete assembly andelectrochemically-cleaned TNB and top layer electrodes, were rinsedthree times with 10 L Tris for 10 s and dried with Ar. The drop size ofPAP_(p) solution placed over the microcavity (inside an Ar-filledglovebag) was 200 nL, and the time for enzymatic conversion varied from30 s to 2 min. The exact volumes and times for specific experiments aredescribed in the text.

Surface characterization. The various stages of surface modification onAu macrochips were studied using polarization-modulation Fouriertransform infrared reflectance absorption spectroscopy (PM-FTIR) with aMattson Instruments Research Series 1 instrument. The IR beam wasfocused onto the sample at an incident angle of 77°. The beam wasp-polarized and passed through a ZnSe Series II photoelastic modulator(Hinds) operating at 37 kHz before reaching the cooled HgCdTe detector.Spectra were taken with 4 cm⁻¹ resolution and a half-wavenumber of 2900cm⁻¹. PM-FTIR spectra were normalized by fitting the differentialreflectance spectrum between 4000 cm⁻¹ and 2100 cm⁻¹ and between 2500cm⁻¹ and 800 cm⁻¹ to 3rd order polynomial backgrounds using FitIT curvefitting software (Mattson). After curve fitting, the spectra weretruncated and converted to absorbance using a WinFirst macro, writtenin-house under the specifications of Mattson. The sample chamber waspurged with dry CO²-free air from Balston air dryer (Balston, Inc.,Haverhill, Mass.). Each modified chip was kept in a vial filled with Arprior to PM-FTIR analysis.

Electrochemical measurements. A BAS-100B potentiostat and a PA-1preamplifier with BAS-100W electrochemical software (BioanalyticalSystems, Lafayette, Ind.) were used to perform CV. A Low Current Moduleand Faraday cage were used for electrochemical experiments on allmicrocavity devices. All electrochemical experiments involving a smalldrop of solution on the microcavity involved placing both the device anddrop in a petri plate containing water droplets and cotton tips soakedin water to minimize evaporation. Two additional open petri platescontaining water were placed inside the Faraday cage to keep the airhumid.

Initial electrochemical characterization of all electrodes was performedin a solution containing 4 mM K₃F_(e)(CN)₆, 1 mM C_(a)Cl₂, and 0.1 MKCl. When the Au macrochips and top layer Au of the microcavity deviceswere characterized, a Pt flag auxiliary electrode and Ag/AgCl (saturatedKCl) reference electrode were used. When the RMD and TNB electrodes ofthe microcavity devices were characterized, an internal setup was used,where the top layer Au served as a combination auxiliary/pseudoreferenceelectrode.

Immunoassay activity of modified Au and PI macrochips was determined byevaluating the surrounding PAP_(p) solution electrochemically for thepresence of PAP_(r) using an external setup of a bare Au macrochipworking electrode, a Pt flag auxiliary electrode, and a Ag/AgCl(saturated KCl) reference electrode. Working electrode potentials werekept within appropriate ranges to avoid electrochemical conversion ofPAP_(p) into PAP_(r). The Au underlying the modifying layer was neverused to detect the enzymatically-generated PAP_(r).

In the small volume, self-contained, electrochemical immunoassay studiesusing the microcavity device, immunoassay activity at the modified RMDwas determined by evaluating the 200 nL drop of PAP_(p) solutionelectrochemically for the presence of PAP_(r) using an internal setup,where the TNB served as the working electrode and the top layerfunctioned as a combination auxiliary/pseudoreference electrode. The RMDunderlying the modifying layer was never used to detect theenzymatically-generated PAP_(r).

Modification and characterization of gold macrosubstrates. Studies usingSAMs of MUA and MUOL for immobilization of protein and DNA have beenpreviously reported. However, to our knowledge, this is the first reportof using MUA and MUOL SAMs for immobilization of rat-anti mouse IgG togold surfaces in a sandwich-type ELISA for detection of mouse IgG.Consequently, we performed several characterization and activity studiesof the modified surfaces. Previously reported studies have used thiocticacid and cysteamine for immobilization of anti human IgE onpiezoelectric quartz crystal with gold electrodes. Thioctic acid SAMshave been used for the detection of mouse IgG1 and rabbit IgG.Butanethiol SAMs have been used for rabbit IgG detection.Photoimmobilization of mouse IgG on Au has been accomplished using SAMsof 10,10′-dithiobis(decanoic acid N-hydroxysuccinimide ester) terminatedalkyl disulfide. Previous studies have used various SAMs to attachproteins other than IgG.

Each stage of modification (SAM, Ab, Ag, and AP−Ab) exhibited acorresponding increase in absorption in the vibrational modes of boththe C—H stretching ( ) region 90-93(CH3 as 2960 cm-1, CH3 sy 2870 cm-1,CH2 as 2920 cm-1, CH2, sy 2855 cm-1) and the amide region (C═O amide Iat 1675 cm-1, N—H amide II at 1545 cm-1, and C—N amide III at 1445 cm-1where ìasî is asymmetric and ìsyî is symmetric.

Immunoactivity of modified surfaces and the role of self-assembledmonolayers was investigated by detecting the enzymatically-generatedPAP_(R) at a nearby bare electrode. The solid and dashed curves in FIG.13 show typical CV responses using an external electrode setup in asolution of 4 mM PAP_(p) and 0.1 M Tris, in which a modified Aumacrochip surface (SAM+complete assembly) had been soaked for 24 h.Higher currents were obtained when the SAM component of the modifiedsurface was prepared with MUOL than with MUA.

When immunoactivity was investigated at bare Au macrochips that had beenexposed to all of the steps in the complete assembly but without the SAMcomponent, no PAP_(R) was detected. The CV response is shown in FIG. 13(dotted curve). Nevertheless, PM-FTIR shows significant amounts ofphysisorption of the individual components of the immunoassay to the Aumacrochip in the absence of the SAMs (data not shown). These resultssuggest that the SAMs are necessary to maintain the active conformationsof the immunoassay components on the gold. In addition, physisorption,if any, to the silicon dioxide on the back side of the chip must notcontribute to the generation of PAP_(R). In fact, silicon wafermacrochips that were subjected to the same surface modification steps asAu-coated macrochips showed no electrochemical activity. The specificactivity at SAM sites is a useful phenomenon, because it can be used tofacilitate the construction of arrays of multi-analyte microassays.

Elimination of physisorption of active species on polyimide. Beforetransferring the surface modification procedure established for the Aumacrochip to the RMD of the microcavity, the adsorption of immunoassaycomponents to polyimide was studied. Polyimide forms the 4 μm thickinsulator between metal layers and essentially serves as the mainmaterial along the walls of each microcavity. Because the desiredimmunoassay configuration involves a selectively-modified RMD usingelectrochemistry to control the site of modification, it would becounterproductive if immunoassay components were to physisorbuncontrollably in their active forms to the surrounding walls.

Polyimide-coated Au macrochips were subjected to the same surfacemodification processes as the Au macrochips. Note that no Au is exposedon the PI coated chips so that chemistry is confined to the PI on oneside of the substrate and silicon dioxide on the other side. Themacrochips were then placed in a PAP_(p) solution for 24 h and CV wasobtained at a gold electrode in an external setup arrangement to detectPAP_(R). The complete assembly with and without the MUOL or MUAexhibited significant generation of PAP_(R), thereby, indicating thatthe immunoassay components physisorbed in an active form on thepolyimide.

Several strategies were investigated to eliminate the activity on thePI. One approach involved first exposing the PI to acetate TBSA solutionin order to block possible protein adsorption sites before proceedingwith the immunoassay assembly decreases in the PAP_(R) current. Assumingthe residual activity to be caused by covalent attachment of the Ab to—NH₂, —OH, and —COOH functional groups of amino acids of BSA, steps weretaken to protect these sites covalently by subsequently exposingsubstrates to EDC and butanol and propionic acid. This resulted infurther decrease but not total elimination of the PAP_(R) signal. Thus,it seems that PI may have multiple sites of physisorption, which havedifferent affinities for different proteins, or that the size of BSAmight prevent complete coverage of those sites.

The second approach to passivate PI activity toward the immunoassaycomponents involved attempts to form more hydrophobic sites on the PI,which should change the nature of the protein adsorption. Thus, the PIwas exposed to small molecules of a more hydrophobic nature (MH, MOD,and DPA). Pretreatment of the PI macrochips with DPA, MH, MOD, and amixture of MH and MOD in ethanol showed a 50% decrease in signal but didnot completely eliminate the activity of the immunoassay A combinationof acetate TBSA with 4 mM MOD completely passivated the PI. At presentwe do not know why this process provides successful elimination of theimmunoactivity and the others do not. The acetate TBSA+MOD pretreatmentwas chosen for use with microcavity devices to prevent physisorption ofactive immunocomponents on the PI.

Electrochemical removal of passivating layers at gold surfaces. Not onlydoes soaking the microcavity devices in acetate TBSA with 4 mM. MODpassivate the PI, but it also passivates the RMD, TNB, and top layer Au.This is an advantage, because it essentially protects the electrodesurfaces from fouling during the immunoassay assembly process.Electrochemical desorption, was performed to remove passivationspecifically at the RMD. CV in F_(e)(CN)⁶³⁻ solution, demonstrates thatpassivation at the RMD is removed but not at the TNB and the top layergold. The clean RMD was then subjected to the procedure for assemblingthe SAMs and the immunocomponents (Ab+Ag+AP−Ab).

Only after deposition of the MUOL or MUA SAMs with the complete assemblyat the RMD were the passivating layers on the top layer Au and the TNBremoved using electrochemical desorption. The cleaned TNB and the toplayer Au could then serve as working and pseudoreference/auxiliaryelectrodes, respectively, to detect PAP_(R), enzymatically-generated atthe modified RMD upon addition of PAP_(p) solution to the microcavity.

Site-specific, self-contained, small volume microelectrochemicalimmunoassay in a microcavity. In a 50-μm diameter microcavity wheresteps had been taken to eliminate the physisorption at the PI, thesurface of the RMD was modified with MUOL+Ab+Ag+AP−Ab (see FIG. 4), andthe top layer of Au and the TNB were electrochemically cleaned. The TNBserved as the working electrode and the top layer Au served as thepseudoreference/auxiliary electrode. The volumes of solutions containingAg and AP−Ab that were used during the assembly were 1 μL. Forcomparison, the smallest sample volume (which is not allowed to dry) forelectrochemical ELISAs reported in the literature and commerciallyavailable for mouse IgG is 10 μL. Thus, our sample volume exhibits atleast a 10 fold improvement. CV responses at increasing time incrementsto a 200 nL drop of the PAP_(p) solution that was placed on top of themodified microcavity are shown in FIG. 14. The volumes of PAP_(p)solution previously reported in the literature are 20 μL or larger.Hence, our system has accomplished enzyme substrate volume reduction bytwo orders of magnitude. Even smaller volumes with our system arepossible (16 pL for the 50-μm diameter cavity and 0.6 pL for the 10-μmdiameter cavity), because working and auxiliary/reference electrodes arelocated within the microcavity. But because of complications with smallsample manipulations and evaporation issues, we only report volumes of200 nL here. Future work will address the smaller volumes.

At only 30 s after the drop of PAP_(p) solution was placed on top of themodified microcavity, a measurable current of theenzymatically-generated PAP_(R) was recorded at a scan rate of 50 mV/s(FIG. 14). This quick, measurable response is due to the short distancebetween the closely spaced working electrode and modified surface(resulting in steep concentration gradients) and to the geometry(PAP_(R) can only escape from the microcavity by passing by the TNB,although collection efficiency is not 100%). This is a significantimprovement over the prior art (excluding SECM), which requireincubating the modified electrode in PAP_(p) or PNP_(p) for 5 to 30 minbefore performing electrochemical detection. Subsequent responsesexhibit increasing plateau currents due to continuous enzymaticgeneration of PAP_(R). Because detection of PAP_(R) in our system occursalmost immediately after placing the drop on the microcavity, thefraction of PAP_(R) that is present from non-enzymatic hydrolysis, whichcould add an unknown background signal that increases exponentially attimes beyond 20 min, 47 is minimal. The self-containedmicroelectrochemical immunoassay experiments also eliminate the need fortransfer of solutions because enzymatic generation and detection arecarried out in the same space. The total assay time, starting with theaddition of Ag solution to the modified cavity and ending when PAP_(R)is detected, (excluding the electrode-cleaning steps) is 24 min. Forcomparison, commercial ELISA for mouse IgG has a total assay time (overthe same steps) of 1.5-3 h.

Using a microcavity with surface modification containingMUOL+Ab+Ag+AP−Ab at the RMD, the TNB as working electrode and the toplayer Au as the reference and counter electrodes, the timed CV responseto 0.5 L (and 0.2 L, not shown) of 4 mM PAP_(p) placed on top of themodified microcavity is illustrated in FIG. 15. The volume of PAP_(p)reported in previous literature was 20 L or higher. Hence, our systemhas accomplished enzyme substrate volume reduction by two orders ofmagnitude.

About five seconds after the drop of 4 mM PAP_(p) was placed on top ofthe microcavity, a current reading was recorded at a scan rate of 50mV/s that served as the initial response. A second response was recordedafter 45 s that indicated a significant increase from the initialresponse. Subsequent responses are shown in FIG. 15. Previous studieshave reported incubating the modified electrode in PAP_(p) or PNP_(p)for 5 to 30 min before performing the potential scan. Our system allowsperformance of the potential scan right after the drop is placed on themicrocavity. This eliminates the generation of PAP_(R) fromnon-enzymatic hydrolysis that adds an unknown background signal thatincreases exponentially at times beyond 20 min.

The data in FIG. 15 indicates that the self-contained electrochemistryinside the microcavity has been harnessed to eliminate the need for anexternal reference and counter electrode in an electrochemicalimmunoassay. At the same time, the biological component of theimmunoassay is contained at the bottom of the same cavity, therebyeliminating the need to transfer the solution as previously reported.

To determine if modification at the RMD was the only site ofimmunoactivity in the microcavity, the RMD of an activemicroelectrochemical immunosensor was subjected to selectiveelectrochemical cleaning to remove the adsorbed substances. The resultsbefore and after removal of immunoassay components at the RMD are shownin FIG. 16, and confirm that immunoactivity is absent from the polyimidewalls, the TNB, and the top layer of gold. This demonstrates that the PIpassivation chemistry is successful, and that subsequent surfacechemistry can localize the immunoactivity of these devices.

Sensitivity and detection limits of the microelectrochemicalimmunoassay. The performance of a microcavity for detection of PAP_(R)was evaluated via a calibration curve. This was done by monitoring theCV responses after placing a 200 nL drop of PAP_(R) solution ofdifferent concentrations ranging from 5.00 mM to 3.98 mM directly on a50-μm diameter cavity. The current (average of two measurements) islinear with concentration and is shown in FIG. 17. A detection limit of4.4 nM or 880 fmol (or 128 pg) was calculated using the typicalequations at the 99%+ confidence level (t is ˜3 and there are 16 degreesof freedom), where the slope from the calibration curve is 19.7 0.8nA/mM, and the standard deviation from the blank signal (17measurements) from a 200 nL drop of 0.1 M Tris is 29×10-6 nA.

The predicted slope for the calibration curve at the TNB in PAP_(R)solutions is 8.5 nA/mM, based on the analytic expression for thetheoretical diffusion-limited current at an in-plane band electrode atall times (when either radial, planar, or both forms of diffusioncontribute). The value of time used in this calculation is the time ittakes to sweep from the E1/2 value to the potential at which the currentwas measured on the reducing side of E1/2. The diffusion coefficient ofPAP used is 0.79×10-5 cm2s-1.99. The equation may be adapted to thetubular band geometry in an infinitely long tube as long as thediameter-to-electrode width ratio exceeds 100. The average current for a4 mM solution from the two sets of experiments used to obtain thecalibration curve for PAP_(R) in FIG. 17 is 80.9 nA, which is muchlarger than the theoretical value, 34 nA. This may be due to a largerTNB electrode area than expected (some undercutting may occur during theetching process), and access to redox species in a larger volume ofsolution once the diffusion layer exceeds the confines of themicrocavity, and redox equilibrium with bulk solution species throughthe top metal layer. Current did vary from device to device and from onefabrication batch to another. For example, on average across manydevices (n=12), the CV plateau current for a 4 mM PAP_(R) solution is 54nA with a standard deviation of 18 nA.

The low detection limit that was obtained in studies of PAP_(R)solutions at a bare microcavity suggest that low detection limits of IgGat modified microcavities may be possible from theenzymatically-generated PAP_(R). A calibration curve for mouse IgG (FIG.18) was obtained from CV responses (50 mV s-1) to PAP_(r) generatedafter 2 min in a 200 nL drop of 4 mM PAP_(p) in 0.1 M Tris (pH 9.0) attwo different microelectrochemical immunosensors for each of fourdifferent concentrations of IgG (5, 10, 50, and 100 ng/ml). Eachimmunosensor was prepared using a 1 mL drop of IgG solution. Because adifferent microcavity was involved for each concentration, it wasnecessary to normalize the immunosensor response. Theenzymatically-generated response at each modified microcavity in 4 mMPAP_(p) solution was divided by the response under similar conditions atthe bare microcavity (before modification) in 4 mM PAP_(R). Theresulting number represents the normalized signal, which should be lessthan or equal to one. A least squares line through the points in thecalibration curve produces a slope of 0.165±0.007 mL/ng. The detectionlimit, using the slope from this line, was determined to be 56 fM (9pg/mL) or 9 fg (56 zmol) of mouse IgG on a 1 μL drop.

The effect of evaporation vs. enzymatic generation. The concentration ofspecies in the small volumes used throughout the immunoassay studieswill change significantly due to evaporation of solvent if precautionsare not taken. All steps involving small drops were performed in asealed, water-saturated environment, with the exception of thoseinvolving the PAP_(p) drop and electrochemical analysis. In the lattercase, evaporation still plays a role to some extent, because the humidenvironment was not completely sealed due to an opening thataccommodated the edge connector leads. In order to identify the timelimit within which electrochemical analysis on 200 nL PAP_(P) solutionsshould be completed, studies on the effect of evaporation wereperformed.

Because current is proportional to the localized concentration at thedetecting electrode, the plateau current of the CV responses of asolution containing a redox species can be used to follow theconcentrating effect. The CV plateau current for PAP_(R) changes withtime at a modified microcavity and at a bare microcavity. That for thebare microcavity is constant for about 3 min. After 3 min, andespecially noticeable at 5 min, the current rises, presumably due to theconcentrating effect of evaporation. There is also a noticeable increaseafter 3 min, and especially at 5 min for the enzymatically-generatedPAP_(R) at the modified microcavity. Consequently, the increase incurrent in the first 3 min at the modified microcavity must be due tothe turnover of PAP_(p) to PAP_(R) by the enzyme and not due toevaporation of water. Therefore, times less than 3 min should beselected when using 200 nL volumes under our conditions, so thatdetermination of detection limit and sensitivity is accurate. The 200 nLdrops evaluated at 2 min should be well inside this evaporation limit.Larger drops should show slower concentrating effects due toevaporation.

The PAP_(R) concentration builds up near the detecting electrode. The CVresponse obtained in the 4 mM PAP_(R) solution at the bare microcavityis that which would be expected if all of the 4 mM PAP_(p) solution wereconverted to PAP_(R) by the immobilized alkaline phosphatase at themodified microcavity. After 3 min, the current at the modifiedmicrocavity is about 2.15% of that of the bare microcavity based on thesignal generated by two chips modified with 100 ng/mL Ag. This leads usto believe that even smaller volumes of PAP_(p), assuming evaporation isnot an issue, should provide even faster increases in signal due toPAP_(R) production, because the loss due to diffusion outside themicrocavity is minimized.

Studies on the effect of evaporation were performed on 0.5 L solution ofPAP_(R) in a humid environment. The CV signal changes with time at amodified microcavity and at a bare microcavity. That for the baremicrocavity is constant over about 5 min. After 5 min, the currentbegins to rise, presumably due to concentration of the PAP_(R). Thepercent rise in current after 5 min at the modified microcavity and thebare microcavity are the same. Consequently, the increase in current inthe first 5 min at the modified microcavity must be due to the turnoverof PAP_(p) to PAP_(R) by the enzyme and not due to evaporation of water.Therefore, results for detection limit and sensitivity at small volumesat microcavity devices for times near 5 min or less are not influencedsignificantly by evaporation.

The PAP_(R) concentration builds up near the detecting electrode. Afteronly 5 min, about 1/10 of the total concentration of PAP_(p) can bedetected in the form of PAP_(R), providing a significant signal. Smallervolumes of PAP_(p) should provide even faster increases in signal due toPAP_(R) production, because the loss due to diffusion outside themicrocavity is minimized.

EXAMPLE 2

The following novel technique was designed because there is no existingelectrochemical immunoassay for Cryptosporidium parvum-ZPA. Although themethod described below is macroscale, those skilled in the art willrecognize that it may easily be scaled down to operate within amicrostructure as shown in FIG. 10 and may be adapted for detection of avariety of microorganisms simply by changing the antibodies used.

1) Deposit 50 Å(angstrom) chromium (Cr) followed by 1000 Å gold (Au) on5 on oxidized silicon wafers using thermal vapor deposition.

2) Using a diamond scribe cut the wafer into 1.2×1.2 cm² Au chips.

3) Clean with piranha (3 parts 30% H₂O₂ hydrogen peroxide: 7 partsconcentrated H₂SO₄ sulfuric acid) and rinse thoroughly with running dIwater.

4) Deposit self assembled monolayers (SAMs) using 25 mL of 4 mM MUA(mercaptoundecanoic acid) or MUOL (mercaptoundecanol) in ethanolovernight inside an Ar (argon) purged glove bag.

5) Rinse thoroughly with ethanol. Rinse with deionized (dI) water.

6) Soak in 1 mL of 48 mg/mL Cryptosporidium parvum Ab (IgM) dissolved inPBS (phosphate buffered saline), pH 6.0 containing 0.1 M EDC(1-ethyl-3-[3-(dimethylaminopropyl)]-carbodiimide hydrochloride) for atleast 2 h. Rinse two times with 1 M NaCl (sodium chloride) followed by0.02 PBS, pH 7.4 3×. Rinse with deionized water.

7) Soak in 1 mL PBS-BSA-GS (phosphate buffered saline-bovine serumalbumin-goat serum) overnight inside the refrigerator.

8) Outside the glovebag soak in 1 mL Cryptosporidium oocysts (previouslyheat shocked at 43° C. for 10 minutes) diluted with the 0.02 PBS-BSA-GS,pH 7.4 to desired concentration from a stock of 10⁶ oocyts/4 mL for 6 h.Rinse with PBS-BSA-GS 3×. Rinse two times with acetate TBSA (tweenbovine serum albumin), pH 5.0. Rinse with dI water.

9) Soak in 1 mL alkaline phosphatase labeled antibody to Cryptosporidiumparvum oocysts (10 mL of synthesized Ab−AP in 1 mL 0.02 PBS-BSA-GS) for4 h. Rinse 3× with PBS-BSA-GS. Rinse with acetate TBSA 2×. Rinse with dIwater.

10) Inside the glovebag, soak in 5 mL of Ar purged 4 mM para-aminophenylphosphate (PAPP) in 0.1 M Tris, pH 9.0 inside a capped beaker wrapped inAl foil overnight (ON) inside an Ar purged glovebag.

11) Remove the modified chip, Ar purge the PAPP for 20 to 30 min thenrun a cyclic voltammetry (CV) using a clean Au macro chip as workingelectrode (WE), Ag/AgCl in saturated KCl as reference and platinum (Pt)flag as counter electrodes.

PBS-BSA-GS: 0.02 M PBS (phosphate buffered saline), pH 7.4 with 1%Bovine serum albumin, 10% goat serum, and 0.02% sodium azide.

EXAMPLE 3

The novel DNA-hybridization Assay for Cryptosporidium parvum-Macrosystem described below was developed to demonstrate the feasibility ofthe viability assay for microorganisms. Those skilled in the art willappreciate that this method is easily scaled down to work inside amicrostructure, as shown in FIG. 11B.

Make sure all solutions and solvents are filtered with 0.2 μm filter.Vortex all solutions before to use. Autoclave deionized (dI) water forall solutions. Use only RNAse and DNAase free reagents withoutautoclaving.

Macro Chips

1) Deposit ˜100 Å Cr/1000 Å Au on 5 in silicon wafer (Si/SiO₂) using BOCEdwards Thermal Evaporator.

2) Dice with a diamond tip scrib to 1.2×1.2 cm² pieces or macro chips.

3) Soak macro chips in piranha solution for 30 min then wash thoroughlywith running dl water.

MUOL SAMs

1) Inside an AR filled glovebag, soak the chips in 4 mM MUOL in ethanolovernight.

2) Rinse three times with ethanol followed by dI water.

DNA Probe 1 (P₁)

1) Place the MUOL modified macro chip in a solution containing 5 mLsolution of Probe 1 in 1 mL of 0.2 M EDC in 0.1 M PBS, pH 6.0.

2) Incubate at room temperature (RT) or 41° C. for 0.5 to 3 h.

3) Rinse 3× in 2×SSC (sodium chloride sodium citrate buffer) using 5 mLeach for 5 mm.

4) Rinse with 1×SSC then soak in 20×SSC for at lest 0.5 h.

Hybridization of Target DNA (T)

1) Place 5 mL T in 1 mL 20×SSC.

2) Soak macro chips previously modified with MUOL+P1.

3) Incubate for 0.5 to 3 h at RT or 41° C.

4) Rinse 3 times in 2×SSC using 5 mL each for 5 min.

5) Rinse with 1×SSC then soak in 20×SSC for at lest 0.5 h.

Hybridization of DNA Probe 2 (P₂)

1) Add 5 mL P₂ in 1 mL 20×SSC.

2) Soak macro chips previously modified with MUOL+P₁+T

3) Incubate for 0.5 to 3 h at RT or 41° C.

4) Rinse 3 times in 2×SSC using 5 mL each for 5 min.

5) Rinse with 1×SSC then rinse with filtered 0.1 M Tris, pH 9.0.

Bioassay

1) Soak the SAMs+P₁+T+P₂−AP modified macro chip in 5 mL of 4 mM PAPP inan Aluminum foil wrapped beaker with a cap overnight in an Ar-saturatedglovebag.

2) Using a clean bare Au macro chip as working electrode, platinum flagas counter electrode, and Ag/AgCl (saturated KCl) as referenceelectrode, perform cyclic voltammetry on the solution surrounding themodified macro chip at 50 mVs⁻¹.

Whereas, the present invention has been described in relation to thedrawings attached hereto, it should be understood that other and furthermodifications, apart from those shown or suggested herein, may be madewithin the spirit and scope of this invention.

1. A method for detecting chemical compounds, said method comprising thesteps of: addition of a sample to immunomicrobeads; allowing an analytein said sample to bind to an analyte-binding material on a surface ofsaid immunomicrobeads; rinsing said immunomicrobeads such that saidanalyte remains bound to said analyte-binding material while theremainder of said sample is removed; incorporating said immunomicrobeadsinto a microassay structure, said microassay structure comprising atleast one three-dimensional microstructure having a substrate andalternating conducting layers and insulating layers, wherein saidconducting layers comprise a plurality of independently addressableelectrodes sandwiched between said insulating layers, and wherein saidmicroassay structure includes a material that facilitatesphysiabsorbtion of said immunomicrobeads; allowing said analyte-bindingmaterial to attach to one of said conducting layers or said insulatinglayers; addition of a secondary analyte-binding material having anelectroactive complex attached, said electroactive complex having anelectroactive species capable of generating an electric current and/orvoltage by either accepting or transmitting one or more electrons to atleast one of said electrodes; allowing said secondary analyte-bindingmaterial to bind to said analyte; rinsing said microassay structure suchthat only said secondary analyte-binding material bound to said analyteremain in said microassay structure; activating said electroactivecomplex such that said electroactive species is released from saidelectroactive complex resulting in said electric current and/or voltageto be generated within said microassay structure, wherein said step ofactivating of said electroactive complex is selected from the groupconsisting of change in temperature, change in pH, denaturation oraddition of an activating compound; and measuring said electric currentand/or voltage within said microassay structure.
 2. The method of claim1 wherein said electroactive complex is selected from the groupconsisting of proteins, enzymes, metalloproteins, dendrimers, redoxenzymes, protein-enzyme conjugate, nucleotide-enzyme conjugate orchelating agents.
 3. The method of claim 1 wherein said analyte-bindingmaterial is selected from the group consisting of antibodies,polynucleotides, lipid layers, dendrimer or a protein binding compound.4. The method of claim 1 wherein said analyte-binding material iscomprised of antibodies, polynucleotides, lipid layers, ligands,proteins or a protein binding compound covalently bound to a selfassembled monolayer or a polymer attached to at least one of saidconducting layers or insulating layers.
 5. The method of claim 1 whereinsaid analyte-binding material is specific to said analyte in saidsample.
 6. The method of claim 1 wherein said secondary analyte-bindingmaterial is specific to said analyte bound to said analyte-bindingmaterial.
 7. The method of claim 1 wherein said analyte-binding materialis a plurality of analyte-binding materials and said secondaryanalyte-binding material is a plurality of secondary analyte-bindingmaterials.
 8. The method of claim 7 wherein said plurality of secondaryanalyte-binding materials are added as a mixture in a single solution orindividually in different solutions.
 9. The method of claim 7 whereinsaid electroactive complex is activated individually or as a mixture.10. The method of claim 7 wherein said electric current and said voltagegenerated by said electroactive complex are measured in a simultaneousprocess or as a combined signal.
 11. The method of claim 7 wherein saidplurality of analyte-binding materials are individually attached to atleast one of said conducting layers or said insulating layers of saidmicrostructure.
 12. The method of claim 1 wherein said microstructure isa mirocavity.
 13. The method of claim 12 wherein said microcavity has anopen end and a closed end, such that access to said analyte-bindingmaterial is via said open end.
 14. The method of claim 1 wherein saidinsulating layers are non-conductive and separate the conductive layers.15. The method of claim 1 wherein at least one of said electrodes is abottom layer of said microstructure.
 16. The method of claim 15 whereinsaid analyte binding material is tethered directly to said electrode onsaid bottom layer of said microstructure.
 17. The method of claim 1wherein said alternating conducting layers and insulating layers form atleast one wall of said microstructure.
 18. The method of claim 1 whereinsaid conducting layers comprise materials selected from the groupconsisting of metals and inorganic materials.
 19. The method of claim 1wherein said alternating conducting layers and insulating layers areconstructed using photolithography, vapor deposition, sputtering orelectroplating.
 20. The method of claim 1 wherein said microstructure isa micropore.
 21. The method of claim 1 wherein said substrate comprisesmaterials selected from the group consisting of a silicon wafer,ceramic, glass and a polymer.